Abstract Blood disorders could be cured by transplantation of hematopoietic stem and progenitor cells (HSPCs). However, because of lack of matched donors, many patients cannot undergo curative therapy. Thus, there is an unmet demand for engineering autologous HSPCs. The long-term goal of this project is to enable conversion of readily accessible autologous adult endothelial cells (ECs) into engraftable HSPCs. To enable this, we need to uncover the niche signals that drive the birth of human HSCs. To this end, we have devised a tractable in vitro method to reprogram readily accessible human and mouse adult ECs into hematopoietic cells. In this approach, we expressed four transcription factors (TFs), FOSB, GFI1, Runx1 and SPI1 (FGRS) in adult primary ECs along with coculture with vascular niche, to reprogram adult ECs into engraftable human HSPCs (rEC-HSPCs). While human rEC-HSPCs could engraft NSG mice, we could not assess whether these cells could give rise to functional T cells. Therefore, we isolated adult lung ECs from Runx1-IRES-GFP reporter mouse for conversion and to tract and modulate the emergence of HSPCs. We show that transient FGRS transduction of Runx1-IRES-GFP ECs along with vascular niche induction, over a 28 day period, enable transition through 3 steps of Induction-Specification-Expansion to generate repopulating mouse rEC- HSPCs. Notably, shutting off FGRS on day 28 before transplantation of CD45.2+ rEC-HSPCs was key to enable multi-lineage engraftment in 1o and 2o CD45.1+ lethally irradiated recipients. Engrafted T cells restore immune response in Rag1-/- mice eliciting adaptive immune response. Single cell clonal and limiting dilution transplantation of day 28 rEC-HSPCs show that these cells are capable of clonal expansion and contain 1/557 competitive repopulating units (CRU) thereby subsets of HSPCs represent long-term rEC-HSCs. Thus, we hypothesize that transient expression of FGRS in adult ECs along with induction by vascular and stromal niche signals will enable deciphering the hierarchy of signals that orchestrate accelerated transition of ECs to abundant engraftable long-term immunocompetent human rEC-HSPCs. These approaches set up the stage for generation of human rEC-HSCs. We will test this hypothesis by examining the following specific aims: 1) Employ non-integrative modified RNA (modRNA) delivery of FGRS TFs into adult human ECs to augment the efficiency, efficacy and safety of conversion of adult human ECs into rEC-HSPCs. 2) Identify tissue- specific specialized human vascular and stromal cell niches that orchestrate the induction, specification, and expansion of FGRS-transduced ECs into abundant immunocompetent long-term engraftable human rEC- HSPCs. 3) Decipher the role of human vascular and stromal niche derived signals, including Cxcl12:Cxcr4/Cxcr7 and BMP4/TGF-?1 in stepwise conversion of human adult ECs into rEC-HSPCs.! Collectively, our mechanistic studies will not only identify ontological pathways involved in differentiation of ECs to rEC-HSPCs, but also translate the potential of engineered autologous HSPCs to the clinical setting. !